We have previously reported that gene-expression profiling employing peripheral blood specimens and oligonucleotide microarrays constitutes a high-throughput approach to dissect the biology underlying operational tolerance in human liver transplantation (11
). The current study was designed to determine whether this approach could be employed to identify genomic classifiers that would (a) comprise modest numbers of genes, (b) provide high diagnostic accuracy in the identification of tolerant recipients, and (c) yield reproducible results across different transcriptional platforms. We first analyzed peripheral blood samples obtained from operationally tolerant liver recipients and from nontolerant recipients requiring maintenance immunosuppression employing Affymetrix microarrays. The diagnostic applicability of the resulting 26-probe genetic classifier was tested on an independent cohort of 19 STA recipients. These patients were selected according to the clinical criteria most commonly used to enroll patients in immunosuppressive weaning trials (1
) and are therefore representative of the diversity of patients to whom a diagnostic test based on the identified gene signature would be applied if adopted for broad clinical use. Prediction of tolerance status based on the identified gene signature resulted in the identification of 4 of 19 potentially tolerant recipients (26%), which matches the prevalence of operational tolerance observed in patients selected according to the above clinical criteria (1
). The most informative genes selected in the microarray experiments were then validated on a qPCR platform. This resulted in the identification of 3 qPCR-derived composite models incorporating 2–7 genes exhibiting remarkable accuracy at discriminating TOL from non-TOL samples in both training and independent validation sets. qPCR experiments incorporated an additional group of samples collected from healthy nontransplanted individuals (CONT). This allowed comparison of TOL and CONT expression patterns. While tolerance-related expression signatures resembled CONT more than non-TOL patterns, half of the genes differentially expressed between TOL and non-TOL samples were also significantly different when comparing TOL and CONT samples. This indicates that a substantial proportion of identified genetic classifiers are very likely to be tolerance specific.
The potential impact on tolerance-related gene-expression patterns of clinical variables such as age, time from transplantation, type of immunosuppressive therapy, and HCV status was specifically addressed on the microarray dataset. HCV infection had a striking impact on peripheral blood gene-expression patterns, markedly outweighing the effect of tolerance itself in terms of the number of genes influenced. The effect of HCV infection on the set of genes most strongly associated with tolerance was, however, weak, which explains why the 26-probe microarray signature could correctly identify tolerant recipients regardless of HCV-infection status. Time from transplantation was found to be marginally associated with the PAM-derived 26-probe signature. This is concordant with the clinical observation that liver recipients with a longer posttransplant follow-up are more likely to become operationally tolerant (1
) but clearly does not account for the expression differences between TOL and non-TOL recipients detected in our study population. A significant effect of pharmacological immunosuppression on tolerance-related gene-expression patterns was excluded by the negative result of the globaltest association analysis and by our finding that STA recipients predicted to be tolerant were grouped together with TOL recipients, which suggests that a common expression signature prevails regardless of the use of immunosuppressive drugs. Hence, we provide here a series of robust predictive models containing a strikingly small number of features capable of accurately discriminating between operationally tolerant liver recipients and those requiring ongoing pharmacological immunosuppression on the basis of peripheral blood gene-expression patterns.
The underlying biology of operational tolerance in humans is still largely unknown. In the current work we have conducted a whole genome gene-set analysis to gain unbiased insight into the mechanisms of operational tolerance following liver transplantation (see Supplemental Data). This analysis has revealed that the expression signature associated with operational liver allograft tolerance is mainly characterized by enrichment in genes encoding for a variety of NK cell–surface receptors expressed by NK, CD8+
, and γδTCR+
T cells. The influence of NK and γδTCR+
T cells on tolerance-related expression patterns has been further confirmed by the demonstration of a significant association between the expression levels of the most informative genes and peripheral blood NK and γδTCR+
T cell frequencies and by the finding that, in TOL recipients, both γδTCR+
and NK cells (together with other PBMC subsets) exhibit unique expression markers. There are 2 main γδTCR+
T cell subsets in human peripheral blood: Vδ1 and Vδ2. In healthy individuals, Vδ2TCR+
T cells largely predominate in peripheral blood (>80%), while Vδ1TCR+
T cells are the major subtype in tissues such as intestine, liver, and spleen (13
). In operationally tolerant liver recipients, in contrast, peripheral blood Vδ1TCR+
T cells expand and typically outnumber Vδ2TCR+
T cells (10
). Our current analysis indicates that Vδ1TCR+
T cells are the only γδTCR+
T cell subset clearly influencing tolerance-related transcriptional signatures. In addition, we provide evidence that peripheral blood Vδ1TCR+
T cells from tolerant liver recipients exhibit unique expression and cell-surface traits that distinguish them from those present in either nontolerant recipients or nontransplanted healthy individuals. Vδ1TCR+
T cells have been reported to exert immunoregulatory functions in a variety of nontransplantation experimental and clinical settings (14
). In liver transplantation, further studies are needed to dissect the functional properties of Vδ1TCR+
T cells and to determine whether these cells have direct suppressive abilities on alloaggressive lymphocytes or act by producing growth factors and repairing tissue damage, as has been shown for the intestinal mucosa (18
On the basis of gene expression and flow cytometry data presented here, it is clear that tolerant liver recipients are distinct not only from recipients requiring maintenance immunosuppression but also from nontransplanted healthy individuals. This suggests that in liver transplantation, achievement of operational tolerance is unlikely to be due to a “reinitialization” of the immune system resulting in recognition of the transplanted graft as “self.” On the contrary, tolerant liver recipients appear to have developed tolerogenic pathways not readily detectable in peripheral blood of healthy individuals but capable of ensuring the protection of the liver allograft.
Functional profiling of human kidney allograft tolerance employing peripheral blood samples has been previously reported by Brouard et al. (23
) utilizing a 2-color cDNA microarray platform (lymphochip) mainly containing immune-related genes (24
). While it would be critical to find common features between operationally tolerant kidney and liver recipients, comparison of both studies is problematic. First, the 2 array platforms employed (lymphochip and Affymetrix U133 Plus 2.0 arrays) have only 4,733 probes in common, with just 543 of them being present in the SAM-derived 2,482-gene list discriminating between TOL and non-TOL liver recipients (data obtained employing the MatchMiner tool; ref. 25
). This number is very low for detailed evaluation of genome-wide transcriptional similitudes, particularly when comparing 2 distant clinical settings and utilizing 2 different expression platforms. Second, the 2 studies analyze different patient groups (i.e., our study is focused on identifying tolerant individuals among STA recipients while Brouard et al. compare tolerant kidney recipients with chronic rejectors). Despite these limitations, a comparison restricted to functional pathway profiles suggests that the mechanisms accounting for operational tolerance in liver transplantation are distinct from those active in kidney recipients. Thus, operationally tolerant kidney recipients appear to be characterized by a state of immune quiescence with marked downregulation of genes involved in lymphocyte trafficking and activation and upregulation of genes responsible for cell-cycle control (23
). In contrast, in operationally tolerant liver recipients, there is a manifest influence on expression patterns of cellular components of the innate immune cells while changes in proinflammatory pathways are barely noticeable except for HCV-positive recipients. Furthermore, a role for B cells in liver allograft tolerance is not supported by either immunophenotyping or gene expression data, in contrast to what has been reported in kidney transplantation (26
In short, our study reveals that measurement of the expression levels of a small set of genes in peripheral blood could be useful to accurately identify liver recipients who are able to accept their grafts in the absence of pharmacological immunosuppression. Validation of our findings in prospective immunosuppression weaning trials would open the door to the possibility of withdrawing immunosuppressive drugs in recipients with high likelihood of being tolerant. Further, functional analysis of expression patterns suggests that molecular pathways involved in the activation and effector function of innate immunity cell types (NK and γδTCR+ T cells) are central to the maintenance of operational tolerance following liver transplantation. Altogether, our work highlights the value of peripheral blood transcriptional profiling in the immune monitoring of liver transplant recipients and provides insight into the pathogenesis of human allograft tolerance.